Treatment of oxyhemoglobin with methylacetimidate results in rather specific amidination of lysine 40 alpha with a resulting increase in oxygen affinity. This effect is most likely mediated by a displacement of the allosteric equilibrium in favor of the high affinity oxy conformation. Amidination of deoxyhemoglobin results in minimal change in oxygen affinity, presumably because of protection of lysine 40 alpha by its participation in a salt bridge. In this project we propose: 1) To continue our study of the amidination of hemoglobin by various monofunctional imidoesters and to correlate the positions of amidination with changes in various functional properties. 2) To consider the effects of structural restraints introduced by cross-linking hemoglobin with bifunctional reagents such as dimethyladiphimidate (DMA). In particular, we will be concerned with the association-dissociation of subunits, the oxydeoxyhemoglobin transition, and the production of polymeric hemoglobin by introduction of intermolecular cross-links. 3) To extend these studies to hemoglobin S in order to correlate the cross-linking of specific residues with the physiological results obtained by other workers studying DMA cross-linking of sickle cells. 4) To study some of the physiological properties of intact erythrocytes after treatment with mono- and bifunctional imidoesters.